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Templated Synthesis of Glycoluril Hexamer and Monofunctionalized Cucurbit[6]uril Derivatives Derick Lucas,† Tsuyoshi Minami,‡ Greg Iannuzzi,† Liping Cao,† James B. Wittenberg,† Pavel Anzenbacher, Jr.,*,‡ and Lyle Isaacs*,† † ‡
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University, Bowling Green, Ohio 43403, United States
bS Supporting Information ABSTRACT: We report that the p-xylylenediammonium ion (11) acts as a template in the cucurbit[n]uril forming reaction that biases the reaction toward the production of methylene bridged glycoluril hexamer (6C) and bis-nor-seco-CB[10]. Hexamer 6C is readily available on the gram scale by a one step synthetic procedure that avoids chromatography. Hexamer 6C undergoes macrocylization with (substituted) phthalaldehydes 12, 14, 15, and 18—in 9 M H2SO4 or concd HCl at room temperature to deliver monofunctionalized CB[6] derivatives 13, 16, 17, and 19—that are poised for further functionalization reactions. The kinetics of the macrocyclization reaction between hexamer and formaldehyde or phthalaldehyde depends on the presence and identity of ammonium ions as templates. p-Xylylenediammonium ion (11) which barely fits inside CB[6] sized cavities acts as a negative template which slows down transformation of 6C and paraformaldehyde into CB[6]. In contrast, 11 and hexanediammonium ion (20) act as a positive template that promotes the macrocyclization reaction between 6C and 12 to deliver (()-21 as a key intermediate along the mechanistic pathway to CB[6] derivatives. Naphthalene-CB[6] derivative 19 which contains both fluorophore and ureidyl CdO metal-ion (e.g., Eu3+) binding sites forms the basis for a fluorescence turn-on assay for suitable ammonium ions (e.g., hexanediammonium ion and histamine).
’ INTRODUCTION The cucurbit[n]uril (CB[n]; n = 5, 6, 7, 8, 10) family of molecular container compounds are prepared by the condensation reaction of glycoluril (1) and formaldehyde under strongly acidic conditions (Chart 1).1,2 Interest in CB[n] molecular containers3 has surged in recent years, in large part due to the high affinity (Ka up to 1015 M1) and highly selective (Krel up to 108 for closely related guests) binding processes that occur inside the CB[n] cavity.47 These large differences in binding free energy amount to a potent driving force that can be used to drive switching processes in biological and technological applications. For example, CB[n] 3 guest complexes have been used in applications ranging from stimuli responsive molecular machines,8 supramolecular polymers,9 sensing ensembles,10,11 and in biomimetic systems.1214 Still other application areas include drug delivery,1517 gas purification,18 and supramolecular catalysis.19 Despite the wide range of applications to which the parent unfunctionalized CB[n] compounds may be applied, there is real need for versatile synthetic methods for the preparation of CB[n] derivatives that contain reactive functional groups that are amenable to further functionalization reactions for incorporation into more complex systems. To date, the most versatile method for the functionalization of CB[n] compounds is the (per)hydroxylation of CB[n] developed by the Kim group,20 who have used the (per)hydroxylated CB[n] compounds for several applications including membrane protein fishing and stimuli responsive r 2011 American Chemical Society
polymer nanocapsules for drug delivery.16,21 Despite these advances, the preparation of monofunctionalized CB[n] compounds containing reactive functional groups remains unknown. For many years, our group has been interested in understanding the mechanism of CB[n] formation (Scheme 1) as a means to prepare new CB[n]-type receptors with the goal of endowing the synthesized CB[n] compounds with exciting new functions.14,22 This line of inquiry has lead us to prepare CB[n] analogues,23 inverted CB[n],24 and nor-seco-CB[n] containers13,25,26 (Chart 1) which were demonstrated to function in applications including UV/vis and fluorescence sensing, chiral recognition, size-dependent homotropic allostery, and foldamer reconfiguration. In the course of these studies, we have garnered mechanistic information that suggested to us that templated synthesis of CB[n]-type compounds would be possible. For example, we established the presence of an equilibrium between S-shaped and C-shaped glycoluril oligomers (e.g., 2C8C)27 that greatly favors the C-shaped form required for the formation of CB[n]-type receptors.28 We also showed that the macrocyclization reaction to give CB[n]type receptors occurs by a combination of chain growth and step growth processes.27,29,30 Previously, Day and co-workers studied the influence of potential templating compounds (e.g., metal ions or ammonium ions) on the product distribution of the CB[n] Received: August 31, 2011 Published: October 04, 2011 17966
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Journal of the American Chemical Society forming reaction.31 Although the presence of templates does effect the ratio of CB[n] formed, the effects are generally modest and the underlying mechanistic reasons for those effects remain unclear. In this paper, we explore the templated synthesis of methylene bridged glycoluril hexamer (6C), the transformation of 6C into monofunctionalized CB[6] derivatives by reaction with (substituted) phthalaldehydes in the presence of templates, and demonstrate unique sensing abilities of a CB[6] derivative covalently functionalized with a naphthalene fluorophore.
’ RESULTS AND DISCUSSION This results and discussion section is subdivided into several subsections detailing the template synthesis of glycoluril hexamer 6C and bis-ns-CB[10], the transformation of 6C into CB[6] derivatives, exploration of the basic recognition properties of these CB[6] derivatives, and their use in creating a turn-on fluorescence sensor for biologically important histamine. Mechanism of CB[n] Formation. First, we review the stateof-the-art understanding of the mechanism of CB[n] formation (Scheme 1) which is required to understand our hypothesis that Chart 1. Structures of Glycoluril (1), CB[n], (()-Bis-norseco-CB[6], and Bis-nor-seco-CB[10]
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templated formation of CB[n]-type receptors will be possible when the reactions are conducted with fewer than 2 equiv of formaldehyde. In brief, the condensation of two molecules of glycoluril (1) with two molecules of formaldehyde delivers a mixture of C-shaped 2C and its S-shaped diastereomer 2S. Previous model system studies have shown that the C-shaped diastereomer is more stable than the S-shaped form by >1.55 kcal mol1, so isomerization to the more stable 2C occurs readily under the reaction conditions.29 Dimer 2C can undergo chain growth by the stepwise addition of glycoluril (1) to deliver trimeroctamer (3C8C); at each step along the way, isomerization from the less stable S-shaped to the more stable C-shaped forms occur. At this stage, two mechanistic pathways may occur. In the first pathway, two molecules of oligomer (e.g., pentamer 5C) may condense with two molecules of formaldehyde via intermediates (()-9(n = 5) and (()-10(n = 5) which leads to bis-nor-secoCB[10] by a step growth process. Similar step growth processes lead to (()-bis-nor-seco-CB[6] and an acyclic glycoluril decamer that we have studied previously.13,26 A second pathway involves the condensation of 6C with formaldehyde to initially deliver (()-9 (n = 6) followed by macrocyclization to give norseco-CB[6]. On the basis of the results of model system studies and product resubmission experiments, we depict all of these fundamental steps with reversible equilibrium arrows.27,32 Finally, the reaction of nor-seco-CB[n] with formaldehyde delivers CB[n]. Within the CB[n] product manifold, resubmission experiments conducted by Day establish that CB[8] undergoes contraction to the smaller CB[n] (n = 5, 6, 7) but that CB[5], CB[6], and CB[7] are stable to the reaction conditions.2 Therefore, CB[5]CB[7] are kinetic traps in the CB[n] forming reaction; the relative thermodynamic stability of CB[5], CB[6], and CB[7] have not been established experimentally. This result implies that the final ring closing transformation of nor-secoCB[n] to CB[n] is irreversible under the reaction conditions. On the basis of this analysis, it is perhaps unsurprising that previous attempts to use ammonium ions that are good guests for CB[n] as thermodynamic templates have not been particularly successful. The key hypothesis explored in this paper is whether substituted ammonium ions (e.g., p-xylylenediammonium ion 11) are
Scheme 1. Mechanism of CB[n] Formationa
a
Color code: reversible equilibria, green arrows; irreversible reactions, red arrows. 17967
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Figure 1. Illustration of the use of 11 as a probe of CB[n] reaction mixtures by 1H NMR spectroscopy.
suitable templates for nor-seco-CB[n] forming reactions conducted between 1 equiv of glycoluril and less than 2 equiv of formaldehyde. Under these formaldehyde deficient reaction conditions, irreversible cyclization to CB[n] is slowed down27 allowing thermodynamic effects to become enhanced. Classification of Templates. Classification of the different types of template effects that may occur in covalent bond forming reactions has been beautifully reviewed by Anderson and Sanders and is therefore only briefly described here.33 Template molecules promote the formation of a specific product by either kinetic or thermodynamic means. Thermodynamic templates enhance the yield of reactions under thermodynamic control by stabilizing one product at the expense of others. Kinetic templates influence the yield of a particular product by changing the various transition state energies and therefore the rate of product formation. Kinetic templates may be further classified as positive or negative based on whether they speed up or slow down the rate of formation of a particular product, respectively. Finally, because kinetic templates, which influence transition state energies, typically bind to reaction intermediates or products, they commonly also exert thermodynamic influences on the reaction. Templated Synthesis of Hexamer 6C and Bis-nor-secoCB[10]. Selection of Para-xylylenediammonium Ion as Template and 1H NMR Probe Guest. On the basis of the hypothesis described above, we decided to conduct reactions between 1 equiv of glycoluril (1) and less than 2 equiv of formaldehyde in the presence of an ammonium ion as template. As the template ammonium ion we selected p-xylylenediammonium ion (11) for several reasons (Figure 1). First, 11 is known to form well-defined hostguest complexes with CB[6], CB[7], CB[8], i-CB[6], i-CB[7], 6C, bis-nor-seco-CB[10], (()-bis-nor-seco-CB[6], and nor-seco-CB[6] which means that 11 could conceivably act as a stabilizing template for many different CB[n]-type receptors. Second, because the kinetics of exchange for complexes of 11 with each of these containers is slow on the NMR time scale, each different container 3 11 complex gives a diagnostic pattern of resonances in the 1H NMR spectrum (Figure 1). For example, for highly symmetric hosts (CB[6], CB[7], i-CB[6], i-CB[7], and 6C), the 1H NMR resonance for the ArH atoms of 11 appears as a sharp singlet in the relatively open 67 ppm region of the 1H NMR spectrum (Figure 1 and Supporting Information). Conversely, for less symmetric bis-nor-seco-CB[10] 3 112 and nor-seco-CB[6] 3 11 complexes, the ArH (Ha and Hb) atoms are nonequivalent and appear as a pair of doublets. Finally, for (()-bis-ns-CB[6] 3 11, the ArH (Ha and Hb) atoms are equivalent and appear as a singlet but the CH2-groups of guest 11 are diastereotopic (Hc and Hc0 ) and appear as a pair of doublets in the upfield region of the spectrum. Third, we find that using 11 as guest tends to result in good dispersion of the host resonances for the less symmetrical host 3 11 complexes. For these reasons, it was
Scheme 2. Templated Synthesis of 6C and Bis-nor-secoCB[10]
particularly efficacious to use 11 as template and simultaneously as an in situ probe for analysis of the content of CB[n] reaction mixtures. Discovery of Templated Synthesis of Hexamer 6C. After much experimentation, we discovered that heating a mixture of 1 (7.1 g, 1 equiv, 5 M), paraformaldehyde (1.67 equiv), and 11 (0.1 equiv) in concentrated HCl at 58 °C for 35 days delivers a thick off-white precipitate that can be isolated by centrifugation.34 Analysis of the 1H NMR spectrum of the precipitate indicates that it contains mainly (≈ 89% purity) the 6C 3 11 complex (Scheme 2). We also analyzed the content of the supernatant by 1H NMR. The supernatant contains 11% 6C, 10% bis-ns-CB[10], 5% nor-seco-CB[6], 5% CB[6], along with unidentified species. To obtain free 6C, we dissolved the crude solid in water, centrifuged away insoluble materials, and then added 5 M aq NaOH which resulted in the precipitation of 6C. Hexamer 6C is obtained as a white powder (0.901 g, 10% yield). Our hypothesis is that the presence of 11 as template served at least two purposes: (1) to bind to 6C and therefore thermodynamically stabilize it, and (2) to cause precipitation which also thermodynamically stabilizes 6C toward further transformation. In an attempt to further optimize this reaction, we changed some key variables (e.g., temperature, acid concentration, equivalents of 11, equivalents of formaldehyde) but were unable to further improve the process. Templated Synthesis of Bis-nor-seco-CB[10]. Previously, we have reported that the reaction of 1 (1.42 g, 1 equiv, 2.5 M) and paraformaldehyde (1.67 equiv) in concd HCl at 50 °C delivers bis-nor-seco-CB[10] as an insoluble precipitate (0.238 g, 15%). We found that this reaction is very sensitive to many variables 17968
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Figure 2. Stereoview of an MMFF minimized model of the 6C 3 11 complex.
including the initial mixing of the solid reagents with the HCl solvent, the nature of the reaction vessel (20 mL scintillation vial preferred), and the nature of the vessel closure (14/20 Suba-Seal attached with copper wire preferred).35 Given the high sensitivity of this reaction, it is perhaps unsurprising that we have been unable to scale-up this reaction to the 10 or 100 g levels; reactions on this scale typically deliver large amounts of CB[6] as product. Since the CB[n] forming reaction is a cyclo-oligomerization reaction27 whose fundamental condensation steps respond to changes in concentration over the millimolar to molar range,32 we reasoned that it would be worthwhile to perform the 11 templated reaction at higher concentrations of 1. For example, when the reaction is conducted using 400 g of 1 with [1] = 4 M, the reaction mixture remains homogeneous and 1H NMR analysis of the crude reaction mixture reveals the presence of bis-ns-CB[10] (11%), 6C (6%), ns-CB[6] (5%), and CB[6] (13%). The more soluble compounds 6C, CB[6], and ns-CB[6] were removed by washing with H2O.36 Decomplexation of bisnor-seco-CB[10] 3 112 by washing with 0.1 M NaOH in MeOH followed by recrystallization from HCl delivered bis-ns-CB[10] (32.6 g, 7% yield). The observation of the enhanced formation of bis-nor-seco-CB[10] (derived from two units of pentamer 5C) at lower concentrations and hexamer 6C at higher concentrations of 1 is consistent with our description of the mechanism of CB[n] formation as a copolymerization process between 1 and formaldehyde which is expected to deliver longer oligomers at higher concentrations according to Le Chatelier’s principle. The Role of Template 11 in CB[n] Forming Reactions. There are many possible roles for template 11 in these reactions (e.g., thermodynamic, kinetic, positive, negative). First, it is known from the literature that 11 binds to 5C (Ka = 1.2 106 M1) and 6C (Ka = 2.2 107 M1) substantially stronger than it does to either CB[6] (Ka = 550 M1) or a tetramethyl derivative of 4C (Ka = 5.6 103 M1).30 This information suggests that the formation of the 6C 3 11 and bis-nor-seco-CB[10] 3 112 complexes thermodynamically stabilize these oligomers. Second, the precipitation of the 6C 3 11 complex suggests that complexation of 6C with 11 changes the maximal solubility of the 6C 3 11 complex (relative to 6C) which in turn results in precipitation when that solubility limit is exceeded. Third, Figure 2 shows an MMFF minimized model of the 6C 3 11 complex. An examination of this complex shows that the presence of guest 11 enforces a geometry in which the reactive NH tips of 6C are held apart from each other.37 This geometrical feature might kinetically disfavor transformation of 6C 3 11 into CB[6] 3 11. Given access to gram-scale quantities of 6C, we decided to study the kinetics of the transformation of 6C and paraformaldehyde into CB[6] in the absence or presence of 11 as template as a means to assess the importance of kinetic stabilization of the 6C 3 11 complex on the reaction. We conducted the reactions in
Figure 3. Plot of mole fraction of CB[6] versus time for reactions between 6C and paraformaldehyde in the absence (•) or presence (9) of 11 as template.
concd HCl at room temperature with [6C] = 200 mM. Figure 3 shows plots of the mole fraction of CB[6] determined by integration of the 1H NMR spectra recorded versus time. After less than 2 h, the untemplated reaction is complete, whereas in the presence of 11 as template, the reaction is only 2% complete. Clearly, the presence of 11 as template substantially slows down the macrocyclization reaction between 6C and formaldehyde to deliver CB[6] as product. Accordingly, it appears that 11 as template serves several roles in influencing the outcome of CB[n] forming reactions. These roles include differential thermodynamic stabilization due to complex formation, controlling the solubility of various components of the reaction mixture through complexation, and finally in influencing the kinetics of the macrocyclization step of the CB[n] forming reaction. Because of the complexity of the analysis of such reaction mixtures using templates other than 11, we have not yet been able to expand our study to the full range of potential templates. Transformation of 6C into Monofunctionalized CB[6] Derivatives. Previously, we have studied the reaction between 2 equiv of N,N0 -dimethylglycoluril with o-phthaldehyde (12) under acidic conditions and observed the dominant formation of S-shaped products.32 However, we reasoned that the reaction between 6C and 12 might be successful because 6C is preorganized to form the C-shaped macrocyclic product (Scheme 3). In the event, we allowed 6C (1 g) to react with 12 in 9 M H2SO4 at room temperature for 36 h. Purification of the crude reaction mixture was easily achieved by a combination of precipitation and washing steps to deliver CB[6] derivative 13 in 72% yield (792 mg). Compound 13 is C2v-symmetric and that is reflected in the simplicity of its 1H NMR spectrum recorded as its 13 3 11 complex (Figure 4a). The most diagnostic resonances in the 1 H NMR spectra are those for host protons Hn (6.89 ppm) as well as Ho and Hp (4.93 and 5.12 ppm) which are upfield shifted due to their proximity to the fused aromatic o-xylylene ring. Similar reactions were conducted between 6C and carboxylic acid or nitro-substituted phthalaldehydes 1438 and 1539 which delivered monofunctionalized CB[6] derivatives 16 and 17 in 56% (641 mg) and 58% (65 mg), respectively.40 Figure 4c,d shows their 1H NMR spectra recorded as their complexes with 11 which reflects the lower Cs-symmetry of these CB[6] derivatives. Finally, we conducted the reaction between 6C (1 g) and 2,3-napthalenedialdehyde 1841 in concd HCl at room temperature and obtained 19 as a precipitate. Simple washing with MeOH delivered 19 in pure form (83%, 0.951 g). Unfortunately, we have not been able to obtain crystals of 13, 16, 17, or 19 that are suitable for X-ray crystallographic structure determination. 17969
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Scheme 3. Synthesis of CB[6] Derivatives 13, 16, 17, and 19
Figure 5. Stereoview of the MMFF minimized structure of 13.
Figure 6. Plot of percent product 13 and (()-21 versus time for the reaction of 6C with 12 (9 M H2SO4, RT): (a) percent 13 in the absence of template (•) or with NH4Cl as template (O), and (b) in the presence of 20 as template (• = percent (()-21; o = percent 13) or 11 as template (9 = percent (()-21; 0 = percent 13).
Template Effects Also Operate During the Reaction between 6C and 12. The observation of intermediates by 1H NMR
Figure 4. 1H NMR spectra recorded (400 MHz, D2O, RT) for: (a) 13 3 11, (b) 19 3 11, (c) 16 3 11, and (d) 17 3 11. Only partial assignments are given in panels c and d because the lower symmetry of 16 and 17 complicates the assignments.
To provide some structural information, we minimized the structure of 13 by MMFF calculations (Figure 5). As is readily apparent, the fusion of the o-xylylene ring to the methylene bridges of the CB[6] skeleton results in an overall ellipsoidal deformation of the cavity along the plane defined by the o-xylylene ring. Similar deformations have been observed for CB[6] derivatives prepared from substituted glycolurils42 and for certain CB[6] 3 guest complexes.43 Since the main reason to prepare CB[6] derivatives is to use their host 3 guest binding properties to enable advanced applications, it is critical that the CB[6] derivatives maintain the high affinity and high selectivity binding interactions typical of the CB[n] family. Given the ellipsoidal deformation described above, we decided that it was necessary to experimentally determine the binding constant of these new CB[6] derivatives toward common guests.
during the reactions between 6C and 12 shown in Scheme 3 suggested to us that it might be possible to observe the influence of templates on the reaction between 6C and 12. First, we monitored the kinetics of the reaction between 6C and 12 in the absence of any template (Figure 6a, •) and observed that the reaction delivered 13 over 240 min. Similarly, when NH4+, which can bind to the ureidyl CdO portals but not the hydrophobic cavities of CB[6] compounds, is used as template, we observe comparable reaction kinetics as the untemplated reaction. The situation is very different when larger diammonium ions 20 and 11 are used as templates. For example, when 6C reacts with 12 in the presence of 20 as template (9 M H2SO4, RT), the reaction very rapidly (
